fluid of multiple
sclerosis patients.
Acta Neurol
Scand 1983;67:
151-63.
10. Cherayil
GD. Sialic
acid and fatty
acid concentrations
in
lymphocytes,
red blood cells and plasma
from patients
with mu!tiple sclerosis.
J Neurol Sci 1984;63:1-10.
11. Merlin
J, Meade
C,J. Relevance
of fatty
acids in multiple
sclerosis. Br Med Bull 1977;33:67-71.
12. Dick G. The aetiology of multiple
sclerosis.
Proc R Soc Med
1976;69:611-5.
13. Hunter MIS, Nlemadim
BC, Davidson DLW. Lipid peroxidation products
and antioxidant
proteins
in plasma
and cerebrospinal fluid from multiple
sclerosis
patients.
Neurochem
Res 1985;10:
1645-52.
14. Rogovina
NI, Koklov
AP. Metabolism
of lipid peroxidation
products
in multiple
sclerosis
patients.
Zh Nevropatol
Psikhiatr
1980;80:696-700.
15. Kilinc A, Yalcin
AS, Yalcin
D, et a!. Increased
erythrocyte
determination
Chem
Ann
Neurol
18. Kurtzke
CLIN. CHEM. 38/12, 2454-2457
in serum
and
1981;8:233-45.
1988;5:113-24.
30. Burton
protocols.
synovial
micro-turbidimetric
fluid
in
for
method
GW, Cheeseman
KH, Ingold KU, Slater TF. Lipid
and products
of lipid peroxidation
as potential
tumour protective agents. Biochem Soc Trans 1983;11:261-2.
31. Huang
Z, Lu F, Lee T. Correlation
between
serum
lipid
peroxides
and thelesion
size in cerebrovascular
disease. Clin Chim
Acta 1988;173:325-30.
32. Esterbauer
H, Koller E, Slee RG, Koater JF. Possible involvement of the lipid peroxidation
product 4-hydroxynonenal
in the
formation
offluorescent
chromolipids.
Biochem J 1986;239:405-9.
33. Al-Timimi
DJ, Dormandy
U. The inhibition
oflipid autoxidation by human
caeruloplasmin.
Biochem J 1977;168:283-8.
(1992)
Reference
Values for Immunoglobulin
Kappa
Kappa/Lambda
Ratio in Children’s
Serum
Maddalena
Saitta,’
Francesco
Aguzzi3’4
Alberto
1987;45:337-51.
28. Weller RO, Swash M, McLellan
DL, Scholtz
CL CliniCal
neuropathology.
New York: Springer-Verlag,
1983:100-1.
29. Oberley LW. Free radicals and diabetes.
Free Rad Biol Med
1983;13:227-31.
JF. Rating neurologic
(peroxidation)
products
arthritis.
J Rheumatol
J, Nishikaze
0. New
lavarone,’
Nasario
Cappello,2
and Lambda
Maria
Rose
Light Chains
Bergami,3
Giovanni
and the
Carlo
Fiorucci,’
We analyzed 708 serum samples from healthy children
and adolescents
by immunonephelometry
to obtain reference values for the immunoglobulin
kappa (ic) and lambda
AddItIonal Keyphrases: pediatric
(A) light chains
methods
for quantifying
immunoglobulin
lambda
(A) light chains
in serum
became
the clinical
laboratory
(1 ), the implications
and
for their
ratio at a time
of life when
immunoglobulin
synthesis
is maturing
and continually
being stimulated.
The A chain concentration
that is to be
maintained
throughout
the child’s life is reached
very
early, just after 1 year, whereas the concentration
ofthe IC
chains, which increases
gradually,
reflects the concentration of the immunoglobulins
as a whole.
values may be useful for studying
illnesses
involving the immune system
K
These
reference
and A chains
in children.
in
immunonephe!ometiy
Since
Laboratory
of Clinical
Chemistry
and Microbiology,
Regina
Margherita
Children’s
Hospital,
Turin,
Italy.
2 Department
ofGenetics,
Biology
and Medical
Chemistry,
Urn1
of Turin,
Italy.
Lab. Analisi,
Ospedale
di Stradella,
1-27049 Stradella,
4 Address
correspondence
to this author.
Received May 26, 1992; accepted August 3, 1992.
3
2454
CLINICAL CHEMISTRY,
Vol. 38, No. 12, 1992
Italy.
.
reliable,
chemistr/
age-related
completely
.
immune
and
status
effects
automated
commercial
kappa
(K) and
available
to
oftheir
ratio
(ic/A) have been widely
studied
in adults.
Most reports
stress
the imbalance
between
the two chains induced
by
the presence
of a monoclonal
component
in monoclonal
gammopathies
(2-4).
The new index, the ratio, seems to
be more useful
in monitoring
than in screening
monoclonal components
with high sensitivity
and specificity
(5-8).
The
versity
Clin
1273-5.
impairment
in MS. Neurology
1983;33:1444-52.
19. Lunec J, Dormandy
TL. Fluorescent
lipid peroxidation
products in synovial
fluid. Clin Sci 1979;56:53-9.
20. Lunec J, Halloran SP, White AG, Dormandy TL. Free radical
oxidation
rheumatoid
21. Iwata
urine.
26. Halliwell
B, Gutteridge
JMC. Free radicals in biology
and
medicine,
2nd ed. Oxford, UK: Clarendon Press, 1989:229.
27. Yagi K. Lipid peroxides
and human
diseases.
Chem Phys
antioxidants
for research
and
23. Knight
JA, McClennan
L, Stahell JK. Cerebrospinal
fluid
lipoperoxides
quantified
by liquid chromatography
and determination ofreference
values.
Clin Chem 1990;36:139-42.
24. Young IS, Trimble ER. Measurement
of malondialdehyde
in
plasma by high performance
liquid chromatography
with fluorimetric detection. Ann Clin Biochem 1991;28:504-.8.
25. Conti M, Morand PC, Levillain
P, Lemonmer
A. Improved
fluorometric determination
ofmalonaldehyde.
Clin Chem 1991;37:
criteria
guidelines
fluid
432-8.
Lipids
sclerosis:
in cerebrospinal
22. Cornbleet
PJ, Gochman
N. Incorrect
least square regression
coefficients
in method
comparison
analysis.
Clin Chem 197925:
susceptibility
to lipid peroxidation
in human
Parkinson’s
disease.
Neurosci
Lett 1988;87:307-10.
16. Dexter DT, Carter CJ, Wells FR, et a!. Basallipid
peroxidation
in substantia
nigra
is increased
in Parkinson’s
disease.
J Neurochem 1989;52:381-9.
17. Poser CM, Paty DW, Scheinberg
L, et a!. New diagnostic
for multiple
of protein
1979;25:1317-9.
1986
IC
and
children
though
ic/A ratio
has been
less studied
Renckens
et al. (9) measured
the
A light
chains
in serum
by immunonephelometry.
the K chain
concentration
in children.
In
concentrations
of
of normal
and diseased
They
noted
that
algradually
increases
with age, the A chain concentration
remains
more
constant
in both diseased
and control
children.
or less
Results
Humoral
immunity
develops
continuously
throughchildhood.
Some pathologies
usually
seen in adults
are now being
seen
in children,
such
as findings
of
monoclonal
components
in cancer
(10) or after organ or
bone marrow
transplantation
(11-13).
Babies and children with congenital
infection
with human
immunodeficiency
virus show the same abnormal
immunoglobulin
synthesis
as do affected
adults
(14-16).
For these reasons
and
others,
we were
interested
in determining
out
these
new
values
immunological
indexes
and
their
Results
ofthe
analysis
ofvariance
in the concentrations
of the ic and
reference
for children.
Materials
and Methods
Subjects
We
evaluated
attending
the
708
subjects,
outpatient
399
boys
department
and
309
girls,
Mar-
of Regina
gherita
Children’s
Hospital
in Thrin
for blood tests
between
May 1989 and June 1991. Ofthe
subjects,
664
were children,
ages 1 month
to 13 years;
and 44 were
adolescents,
ages 14-20 years. All subjects
were apparently
healthy
and well nourished.
From
clinical
data
and interviews
with the subjects’
accompanying
relatives,
who gave
informed
consent
for the collection
of
blood
samples,
we concluded
that the subjects
were free
from acute
immunological
sent to the
infections
system.
laboratory
or chronic
disorders
had
The children
involving
generally
negative.
values
calculated
by parametric
and nonmethods
are shown
in Table
1. To account
for the asymmetrical
distribution
of the population
in
the groups,
we chose the interval
between
the 5th and
95th percentiles
as our reference
range.
For the IC chain,
values from consecutive
age groups
100W#{149}
We quantified
the
K
Beckman
and
A light
Instruments
chains
nephelometrically
(Galway,
Eire)
using
a Beckman
APS
cali-
immunonephelometric
system,
brator
(Cal I, lot M912127)
and antisera.
The serum
samples,
obtained
by centrifugation,
were examined
at
once or frozen
at -20 #{176}C
until
being
tested.
A control
serum
sample
(Beckman
Control
1, lot M980116)
was
inserted
resulting
yielded
9000#{149}
K chain
8OOO
7OOO
-I
6ooo.
5OOO
Immunonephelometry
with
at the beginning
and end of every series; the
total of 70 control
assays
over 18 months
interseries
CVs of 4.7% and 4.3% for K and A,
4OOO
3000
0
,
0.5 1
2
Fig. 1 . Trend of K
adolescence
(mean
,
3
4
and
SE)
5
Values
.
Different
The
two
data
groups
Analysis
were divided
for the first
into
year
14 age groups
for analysis:
of life (0-6 months;
6-12
variance
with
confidence
(18) method.
The
limits
corrected
Student,-Newman-.Keuls
by
the
Bonferrom
(19) multiple-range
test was used to suggest
the possible merging
ofage groups,
which
enabled us to tabulate
reference
values
by age. Statistical
evaluations
were
performed
with the BMPD programs
(20).
12 14 20
childhood
for K and A Chains
Ages
Co,
months),
one group per year up to year 9, and one group
each for ages 10-11,
12-13,
and 14-20
years.
We compared
the averages
for K and A chains
for contiguous
age
groups
and within
each age group by two-way
analysis
of
I
6 7 8 9 O
YEARS
(#{149})
and A (0) light chains during
Table I Reference
respectively.
Statistical
changes
Reference
the
been
displayed
a normal
gamma
zone on serum
IgG,
IgA,
and 1gM were
within
the
(1 7). Tests
for C-reactive
protein
were
that
parametric
to be tested
for asthenia
or pallor
on parental
insistence
or for screening
before surgery
(removal
of tonsils,
urethrocele,
strabismus,
etc.).
All subjects
electrophoresis.
normal
range
show
A light chains
in the
population
examined
are significantly
different
and that
values for each chain are considerably
influenced
by age
(Figure
1). The Bonferroni
test showed
a highly signif
icant
difference
between
values
for the two chains
(P
<0.001)
from
the end of the first year of life on. Thereafter,
the curves
for the two light chains
showed
a
progressive
discrepancy.
The ic chain
concentration
greatly
increased
between
years 1 and 2 (P <0.001).
The
change
was then progressive,
becoming
marked
between ages 4 and 5 years (P <0.05),
until stable values
were reached from year 7 onwards.
By contrast,
A chains,
after
a significant
increase
between
years
1 and 2 (P <0.05),
remained
almost
constant
throughout
childhood
and adolescence.
and
at
mg
th
No. of
Age, yssrs
Kappa
cases
Ms#{149}n
A
2 SD
Mess (2SDs)
rangs
p.rosntll.
rungs
chain
0-1
1-3
3-5
5-7
7-20
Lambda chain
0-1
1-20
66
4010(2620)
1390-6630
1770-6870
104
6140(2640)
3660-8390
131
7060(2940)
3500-8780
4120-10
000
122
8250(2860)
5390-11
285
9090
5370-12
66
642
3280 (2800)
4430 (2000)
2430-6430
CHEMISTRY,
Vol. 38, No. 12, 1992
CLINICAL
(3720)
480-6080
110
810
4950-10 000
5570-10
200
6240-12
300
1690-5730
3000-6130
2455
and children
have become fully capable
of synthesizing
their
own immunoglobulins,
we find an increase
in the
concentrations
of both chains
but a particularly
signi.f.
icant increase
in the K chains.
Apparently,
the maturity
Z2
zo
KLR
of the
1.8
-J
1.6
1.4
1.2
1.0
O’l
I
a
0.5 1
i’o
12 14 20
YEARS
Fig. 2. Trend of ic/A ratio (KLR) during childhood and adolescence
(mean and SE)
Table 2. Reference
Values
for sc/A at Different
idA
Ages
to synthesize
A-type immunoglobulins
is
whereas
the ability to synthesize
K-t3*
develops
more slowly.
The stability
ofthe A chain concentration
until a very
advanced
age was also reported
by Singh
and Kulig
(22), who found that the K chains
continue
to increase,
even in subjects
older than
79 years.
sc/A increases
gradually
with age, paralleling
the increase
in ic chains.
In monoclonal
gammopathies
in adults,
major clinical
information
is obtained
from
the ic/A ratio,
but its
usefulness
in children’s
pathology
is limited.
Nevertheless, it would be ofinterest
to study the behavior
ofIC and
A chains
in the immunological
disorders
ofchildhood.
In
our experience,
toward
5th-95th
psrcsntlls
rungs
Ags,
ysars
No. of
cause
0-1
1-3
3-6
66
104
204
1 .27 (0.62)
1.43 (0.58)
1 .69 (0.62)
0.65-1 .89
0.778-1.87
0.85-2.01
1 .07-2.31
0.969-1.89
1.22-2.51
6-20
334
2.01 (0.84)
1.1 7-2.85
Mssn ± 2
SD rungs
Mssn (250*)
1.42-2.80
for example,
children
of different
ages
with
symptomatic
infection
with
human
immunodeficiency
virus nearly
always
have low sc/A values
(data not
shown),
as ifthe
hyperactivation
of the B-cell compart-
ment
ratio
ability
rapidly
achieved,
immunoglobulins
very
generally
seen
the synthesis
in these subjects was
of A immunoglobulins.
directed
more
We thank the Committee
for Informatic
Systems
of Piedmont,
for use of their computers
for data analysis
and G. Grasso
(Caltagirone
Hospital, Italy)for
sending
samples for analysis.
This
study was partly supported
by funds from the Italian Ministry for
Italy,
University,
Technology
and
Scientific
Research.
References
1. Caron J, Horsfall
K, Penn GM. Utility of K/L quantitation:
ofa multicenter
study [Abstract]. Clin Chem 1986;32:1141.
2. Whicher
JT, Wallage
M, Fifleld R. Use of immunoglobulin
result
overlapped
from year 7 on, according
to the StudentNewman-Keuls
test, which
was sufficient
to suggest
a
grouping
of reference
values
in the 7- to 20-year
range.
For the A chain
we feeljustified
in referring
to only two
groups,
before
and after year
1.
The value for sc/A also increased
progressively
during
childhood
(Figure
2). The significant
increases
occurred
between
years 1 and 2 (Bonferrom
P <0.05),
years
3 and
4 (P <0.05),
and years 6 and 7 (P <0.05).
Relevant
reference
values
are given in Table 2.
Discussion
ic/A ratios
investigated,
ulins
increases
in children
still have not been thoroughly
even though the synthesis
of immunoglobprogressively
during
childhood
because
of the
physiological
maturation
of the B-immune
compartment
and its active stimulation
by antigens.
Moreover,
pathological
conditions
in children
often involve
quantitative
alterations
of immunoglobulin
concentrations, whether
as hypo- or hypergammaglobulinemia.
We determined
reference
values for K and A chains and
their ratio in serum samples
of children
attending
our
outpatient
department,
following
the rules
recommended
for reference
values
in populations
of children
(21).
The trend
we observed
for IC and A chains
overlaps
with
that found by Renckens
et al. (9) in a smaller
number
of cases.
After
the first year of life, when
the
maternally
derived
antibodies
have
been catabolized
2456
CLINICAL CHEMISTRY,
Vol. 38, No. 12, 1992
heavytechniques
and light-chain
Clin Chem
3. Normansell
measurements
compared
of typing
monoclonal
DE. Use of kappa
and lambda
as a means
1987;33:1771-3.
with
existing
immunoglobulins.
chain
quantitation
the detection
of iznmunoglobulin
abnormalities
in serum.
Clin
Immunol
1987;5:100-3.
4. Keren DF, Warren JS, Lowe JB. Strategy to diagnose monoclefor
nal gammopathies
in serum: high-resolution
munofixation,
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Clin
electrophoresis,
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201.
5. Jones RG, Aguzzi F, Bienvenu J, et al. Use of immunoglobulin
heavy-chain
and light-chain
measurements
in a multicenter
trial
to inve8tigate
monoclonal
components:
I. Detection.
Clin Chem
1991;37:1917-21.
6. Jones RG, Aguzzi F, Bienvenu J, et a!. Use of immunoglobulin
heavy-chain
and light-chain
measurements
in a multicenter
trial
to investigate
monoclonal
components:
II. Classification
by use of
computer-based
7. MacNamara
algorithms.
EM, Aguzzi
evaluation
Clin Chem 1991;37:1922-6.
F, Bianchi
P. Assessment
of serial
immunochemical
in myeloma
follow-up
[Abstract].
Biochem
Clin 1989;13(Suppl):314.
8. Aguzzi
F, Bienvenu
J, Jones
R, Whither
J. Monoclonal gemmopathies.
KLR: a new diagnostic
tool. Paris: Pasteur
Diagnostics,
1991.
9. Renckens
AJ.JM,
Jansen
MJH,
van Munster
PJJ, Weemaes
CMR, Bakkeren JAJM. Nephelometry
ofthe kappa/lambda
lightchain
ratio in serum of normal and diseased children. Clin Chem
1986;32:2147-9.
10. Solomon A. Homogeneous
cancer.
11.
(monoclonal)
immunoglobulins
in
Am J Med 1977;63:169-76.
Hammaratrom
L, Smith
CIE.
Frequent
occurrence
of mono-
clonal gammopathies
with an imbalanced
light chain ratio following bone marrow
transplantation.
Transplantation
1987;43:447-9.
12. Mitus
AJ, Stein R, Rappaport
JM, et al. Monoclonal
and
oligoclonal gammopathy
1989;74:2764-8.
afterbone
marrow
transplantation.
Blood
13. Fisher AM, Simon F, Le Deist F, Blanche 5, Griscelli C, Fisher
A. Prospective
study of the occurrence
of monoclonal
gammopathies following
bone marrow transplantation
in young children.
Transplantation
1990;49:731-5.
14. Ng VL, Hwang KM, Reyes GR, et al. High titer anti-HIV
antibody
reactivity
associated
with
a paraprotein
spike
in a
homosexual
male
with
AIDS-related
complex.
Blood
1988;71:
Fudenberg
ml, Wells JV, ode. Basic and clinical immunology.
Los
CA: Lange Medical,
1984:316.
18. Armitage
P. Statistical
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Oxford:
Blackwell
Scientific,
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19. Tukey JW. Exploratory
data analysis.
Reading, MA: Addison
Altos,
Wesley,
1977.
A, Gallo P, Zamarchi
R, et al. IgG oligoclonal bands
in sera of fflV-1 infected
patients
are mainly
directed
against
mv-i
determinants.
AIDS Rae Hum RetrOvirUses
1990;6:581-6.
16. Amadori
A, Chieco-Bianchi
L. B-cell activation
and lilY-i
infection:
deeds and misdeeds. Immunol
Today 1990;11:374-9.
17. Stiehm
ER, Fudenberg
RH. Levels of immunoglobulins
in
20. BMDP statistical
software.
Berkeley: University
of California
Press, 1988.
21. International
Federation
of Clinical
Chemistry.
Provisional
recommendation
on the theory of reference values. Part 2. Selection of individuals
for the production
of reference
values. Clin
Chini Acts 1984;139:205F-13F.
22. Singh J, Kulig K. Measurements
ofkappa
(K) and lambda
(A)
light chains in individuals
of various age [Abstract].
Clin Chem
serum
1990;36:986.
1397-401.
15. Amadori
of normal
subjecth
at different
CLIN. CHEM. 38/12, 247-2464
Spuriously
Ektachem
Generation
ages.
In: Stites
J,
(1992)
Low Concentrations
Slides: Prevalence,
16 Slides
Anick
Kempinaire’
Smeyers-Verbeke,
DP, Stobo
Muriel
Walter
Laureys,’
Cooreman,3
of Serum Iron Measured
with Generation
Possible Causes, and Partial Improvement
Wim Goedhuys,’
Hugo Neels,4
and
Luc Demeulemeester,’
Frans
K. Gorus’5
Claude
14 Kodak
with
Sevens,’
Johanna
For sera with iron (Fe) concentrations
<4 &mol/L, Kodak
Ektachem
slides Generation
(GEN) 14 (without ascorbic
acid) yielded systematically
lower results for Fe than did
liquid Ferrozine-based
reagents
from Baker containing
ascorbic
acid (10 g/L, final concentration)
and adapted to
Cobas-Bio.
Dunng an 8-month comparison
period, outhers (defined
as [Fe],
[Fe]K
>4 mol/L)
were
seen in 21 of the 8731 sera (0.24%) tested, corresponding
dance
of Kodak results with Cobas,
and hence with
flameless
AAS, for both types of outliers;
abolished
Zn
dependency
of results; and increased
Fe results in sera
to <5%
samples
-
of the
addition
types
sera
of ascorbic
of
[Fe]K,,
outliers:
>0.4
with
[Fe],,
<4
mol&.
acid and (or) Fe identified
type
prnol/L,
1
(--70%),
by (supra)normal
In vitro
at least two
characterized
Fe recovery
by
in
Kodak slides in the presence
or absence
of ascorbic acid
(1 0 g/L), and by between-method
differences
in serum Fe
(Cobas
Kodak)
that were significantly correlated with
serum Zn content
(P <0.0004);
and type 2 (--30%),
tentatively ascribed
to contamination
by EDTA, with Serum Fe by Kodak <0.4 mol/L
and Fe recovery
near 0%,
both of which could be significantly and dose-dependently
increased
by addition
of ascorbic
acid (5-20 gIL). For both
types of outliers,
flameless
atomic absorption
spectrometry (AAS)
yielded
results that were significantly
higher
-
than concentrations
by Kodak with GEN I 4. Use of GEN
I 6 slides (containing
ascorbic
acid) improved
concor-
of ‘Clinical
Chemistry,
Academic
Hospital (AZVUB), and oPPharmaceutical
and Biomedical
Analysis,
Pharmaceutical Institute,
Vrije Urnversiteit
Brussel,
Laarbeeklaan
101,
Departments
B-1090 Brussels,
Belgium.
Departments
ofClinical
mistry3
St. Augustinus
Ziekenhuis,
Oosterveldlaan
24, B-2610 Wilrijk, Belgium,
Algemeen
Ziekenhuis Middeiheim,
Lindendreef
1, B-2020 Antwerpen,
Belgium.
5
Author for correspondence.
Received
March
7, 1992;
accepted
August
5, 1992.
with type 2 outliers, although these remained substantially
lower than by Cobas. However,
like other ascorbic acidcontaining
reagents,
GEN
1 6 slides
were
more
sensitive
to interference
by dextran-bound
Fe, as assessed
during
in vitro addition experiments
and comparisons
involving
from
Fe-dextran-treated
patients.
GEN
1 6 slides
are hence expected to more frequently overestimate
the
physiologically
available
protein-bound
Fe in hemodiaiysis patients. In hospital laboratories,
this new interference
will probably arise more frequently than the spuriously low
results with GEN 14, hence warranting further efforts in
optimizing
Fe slides.
Addftional
citrate
zinc
spectmpholometry
Keyphrases:
. divalent
. atomic
oxidants
.
ions . muItiIayer
film
absorption spectrometry
EDTA
.
analysis
We previously
showed
(1 ) that
Kodak
Ektachem
slides [Generation
(GEN) 14; Eastman
Kodak, Rochestsr, NY] for determining
iron (Fe) and total Fe-binding
capacity
in serum
yielded
results
that correlated
very
significantly
with
data obtained
with ascorbic
acidcontaining
Deventer,
centrifugal
liquid
Ferrozine
reagents
(J. T. Baker,
The Netherlands)
adapted
to a Cobas-Bio
analyzer
(Roche
Diagnostics,
Basel,
Switzerland) and with flame atomic
absorption
spectrometry
(AAS;
Instrumentation
Laboratory,
Wilmington,
MA).
Moreover,
Kodak
slides proved superior
to both other
technologies
in that the multilayer
film elements
more
exclusively
measured
the (patho)physiologically
rele-
CLINICAL CHEMISTRY, Vol. 38, No. 12, 1992
2457